Blastomyces dermatitidis is a primary and opportunistic dimorphic fungal pathogen, but little is known about its virulence factors. After B. dermatitidis spores enter the lung, they convert to invasive yeasts. The yeast displays an adhesin WI-I that binds CR3 and CD14 receptors on cells, and evokes strong immune responses. Hypovirulent mutants of the fungus show altered WI-1 expression and adherence. The adhesive and antigenic features of WI-1 highlight its role in host-pathogen interactions. However, a direct role for WI-1 in the pathogenicity of B. dermatitidis has not been firmly established. In the prior grant period, Dr. Klein developed a transformation system for B. dermatitidis, rendering the fungus manipulable on a level that had not been attainable. It is planned to apply that system and other molecular tools developed to directly investigate the role of WI-1 in pathogenicity of B. dermatitidis. In preliminary studies, the laboratory observed that WI-1 is displayed on the yeast rather than on the mold. Dr. Klein hypothesizes that WI-I is expressed preferentially on yeast form cells and enhances their survival within the host by resisting immune defenses or modulating them. Three aims are proposed to address the hypothesis. 1) WI-I expression will be manipulated by targeted gene replacement in North American strains that normally express it, and by gene complementation in African strains that normally do not, to test its pathogenic role in vivo during experimental infection; 2) The cellular and molecular mechanisms whereby WI-1 enhances pathogenicity will be investigated by analyzing and comparing how isogenic strains that do and don't have WI-1 interact with neutrophils and macrophages in vitro; and 3) The preferential expression of WI-1 on yeasts will be studied to delineate how the gene is regulated, using a lacz reporter to map the promoter, and to elucidate how the gene responds to host cues in vivo during infection, using the green fluorescent protein under control of the WI-1 promoter. Results from this work will yield important new information about a putative virulence factor, how it works, and how it's expression is regulated by the fungus and modulated by the host during infection.